1. DNA REPLICATION IN PROKARYOTES

2. DNA replication in prokaryotes
The bacteria multiply by a process of binary fission;
before this occurs, each cell must duplicate its genetic information so that each daughter cell has a copy. By a process called DNA replication
DNA replication:
is the process of producing two identical copies from one origin This biological process occurs in all living organisms and is the basis for biological inheritance.

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4. General Features of DNA replication
DNA replication is an extraordinarily important and complex process upon which all life depends.
DNA replication is semi-conservative
During DNA replication, the two strands of the double helix are separated; each then serves as a template for the synthesis of a complementary strand according to the base-pairing rules.
DNA replication begins at the origin of replication (ori)
DNA is synthesized by the enzyme DNA polymerases
DNA replication is bidirectional from the origin of replication.
DNAreplication is also extremely accurate; E. coli makes errors about one in a million.
Despite its complexity and accuracy, replication is very rapid. In bacteria, replication rates approach 750 to 1,000 base pairs per second.

5. Replication steps
The replication of chromosomal DNA begins at a single point, the origin of replication
DNA replication begins with the unwinding of two anti-parallel complementary strands, resulting in the formation of two single strands.
This unwinding produces the two replication forks. The synthesis of DNA begun at the replication fork.
The replication proceeds in 5’-> 3’ direction and is semi-discontinuous away from the movement of replication fork in short segments called the series of fragments calledOkazaki fragments, this strand called lagging strand.
Of the two strands, one strand is synthesized continuously (5’ to 3’) in the direction of movement of the replication fork called leading strand; while the other strand is synthesized discontinuously.
Two replication forks proceed in opposite directions from the origin until they meet at a site called the replication termination site.

8. DNA replication involves the action of a number of specialised enzymes & proteins :
Helicases: unwinds the ds DNA
DNA topoisomerases: – relieves the topological stress produced in the helical stuctures of DNA during unwinding
DNA polymerase I
DNA polymerase III
DNA primase: is an enzyme involved in the replication of DNA and is a type of RNA polymerase. segment called a primer complementary to a ssDNA template
DNA ligase– catalyses the formation of a phosphodiester linkage.
Single stranded DNA binding proteins – stabilizes the single strands of DNA after unwinding

10. The DNA Polymerase:
DNA polymerase is an enzyme that carries out the synthesis of a new strand on the template strand
Enzymes called DNA polymerases catalyze DNA synthesis.
All known DNA polymerases .
DNA polymerases catalyze the synthesis of DNA in the 5' to 3' direction,
the nucleotide to be added is a deoxynucleoside triphosphate (dNTP ). Deoxynucleotides are linked by phosphodiester bonds formed by a reaction between the hydroxyl group at the 3' end of the growing DNA strand and the phosphate closest to the 5' carbon (the a-phosphate) of the incoming deoxynucleotide

11. The DNA Polymerase Reaction
The DNA Polymerase Reaction. The hydroxyl of the 3' terminal deoxyribose makes a nucleophilic attack on the a-phosphate of the nucleotide substrate (in this example, adenosine attacks cytidine triphosphate).

12. DNA polymerase in E.coli
There are atleast five DNA polymerases associated with E.coli DNA replication
These are
DNA polymerase I – it is the first DNA pol to be isolated and purified. The polymerase has 5’->3’ exonuclease activity
DNA polymerase II – the enzyme is encoded by polB gene. It has no 5’->3’ exonuclease activity, rather it has 3’-5’ exonuclease activity(proofreading activity)
DNA polymerase III – this is the principle replication enzyme,encoded by the gene polC. It has 5’->3’ proofreading activities. It lacks 3’->5’ exonuclease activity. It has a very high proccessivity
DNA polymerase IV and DNA polymerase V are involved in an unusual form of DNA repair

13. DNA Polymerase III
DNA polymerase III holoenzyme is the primary enzyme complex involved in prokaryotic DNA replication
DNA polymerase III synthesizes base pairs at a rate of around 1000 nucleotides per second.
As replication progresses and the replisome moves forward, DNA polymerase III arrives at the RNA primer and begins replicating the DNA, adding onto the 3'OH of the primer
DNA polymerase III will then synthesize a continuous or discontinuous strand of DNA
DNA polymerase III has a high processivity and therefore, synthesizes DNA very quickly.
This high processivity is due in part to the β-clamps that "hold" onto the DNA strands.

14. DNA Replication in Prokaryote (E.coli)
The genome of E.coli is replicated bi-directionally from a single origin, oriC . E. coli replication is circular with no free ends. Replication of DNA in E. coli is also known as theta replication and it occurs in three steps:
Initiation
Elongation
Termination

16. Sequence of events during Initiation
The E. coli oriC , consists of two short sequences:
one is, where DnaA protein binds and the other is called DNA unwinding elements(DUE).
The binding of DnaA facilitates the initial strand separation of E. coli DNA duplex,
DnaC protein binds to the DnaB protein, which results in the opening up of DnaB ring. The two DnaB ring , one on each strand
DnaC is released and two DnaB remains bound to the two ss DNA
DnaB helicase is then loaded on to two ss strands , which then travels along the ss DNA in the 5’->3’ direction unwinding the DNA strand, creating two replication forks.
Unwinding produces stress which is removed by DNA gyrase
Finally the oriC DNA is methylated by the Dam methylase ,

18. ELONGATION
In this phase, the synthesis of two new daughter strand takes place complementary to the template strand
DNA polymerase III is the enzyme that synthesizes the daughter strands
At this point, a primer is needed so that DNA polymerase III can begin to act.
A primer is a short segment of RNA, that provides 3’-OH group to which a nucleotide can be added
This phase is marked by the synthesis of leading strand and lagging strand
Leading strand is synthesized continuously in 5’ to 3’ direction along the direction of the movement of replication fork
Lagging strand synthesis occurs discontinuously by loop formation in short segments called Okazaki fragments.

20. DnaB helicase first unwinds the replication fork
DNA primase then associates with DnaB helicase, which synthesizes a short RNA primer
The primer is then extended by the DNA pol III, which completes the synthesis of one Okazaki fragment
Once an Okazaki fragment has been completed, its RNA primer is removed and replaced with DNA by DNA polymerase I, and the remaining nick is sealed by DNA ligase .
DNA ligase catalyzes the formation of phosphodiester bond

21. TERMINATION
Replication of bacterial genome proceeds bi-directionally which terminates at a position diametrically opposite to the origin of replication.
Replication terminates at the terminus region containing multiple copies of a 20bp sequence called Ter(terminus) sequences
Ter sequence works as the binding site for protein Tus (terminus utilization substance) which stops the DnaB helicase, resulting in termination of DNA replication
The completed chromosomes then partitioned into two daughter cells during cell division